Live, attenuated oral poliovirus vaccine (OPV) has been the primary polio vaccine utilized in developing countries. As we move towards eradication of polio, however, the once-beneficial characteristics of vaccine- related shedding are no longer desirable. Long-term replication of OPV and rapid mutation of the virus can lead to genetically divergent vaccine-derived polioviruses (VDPVs), which can result in paralysis indistinguishable from paralysis caused by wild poliovirus. Thus, one of the current priorities highlighted by the World Health Organization (WHO) are the factors favoring the emergence of VDPVs. WHO currently employs a broad definition of circulating VDPVs (cVDPVs) as strains differing by more than 1 % (serotypes 1 and 3) or 0.6 % (serotype 2) at the VP1 gene, a level of divergence thought to approximate viral circulation for more than one year, and presumed to persist by person-to-person transmission. This definition relies on the assumption that the longer vaccine viruses circulate, the greater the risk they will revert to virulence. However, this definition is based on an outdated estimate of the OPV substitution rate that was estimated from a small sample of 31 isolates of wild poliovirus serotype 1 and may not be representative of the three OPV serotypes. Most importantly, the current definition of a cVDPV is not a measure of epidemiological or genotypic-based risk. Attenuating substitutions in OPV can be lost within a single patient?s infection and a virus 1% genetically divergent from OPV may remain avirulent. As a result, this traditional approach to risk assessment has poor sensitivity and specificity for virulent strains. In order for future polio surveillance to be effective and actionable, an updated definition of cVDPVs is urgently needed. The objective of this proposal is to characterize the early molecular evolution OPV serotypes 1, 2, and 3 after national vaccination campaigns in rural Mexico and provide new tools for identifying pathogenic cVDPVs. We hypothesize that harnessing burgeoning genomic data can yield a tool with accuracy at predicting emergence of pathogenic vaccine-derived viruses. By using Sanger and whole genome sequencing of OPV isolates from time- dated stool samples following an OPV vaccination campaign in Mexico, we aim to estimate a molecular clock for each OPV serotype and to construct a high-resolution phylogenetic portrait of OPV evolution following vaccination campaigns. The Stanford group has long-standing research collaborations with colleagues at the Mexican National Institute of Public Health, recently focused on determinants of household and community OPV shedding and transmission. We will use our published assay for WGS of OPV isolates collected from human samples in Mexico for the proposed studies, which are uniquely suited to help us identify patterns of OPV mutation and variants associated with community circulation, and to help define determinants and genetic markers of development of cVDPVs.